Neuroblastoma, a tumor of the embryonic neural crest, is highly malignant and poorly responsive to conventional therapy, yet tumor cells retain the capacity for neuronal differentiation. This cancer is ultimately fatal in most patients and thus there is an urgent need to identify new agents and treatment strategies. The long-term goal is to understand the relationship between malignancy and cell differentiation state. Cell phenotype and amplification of the N-myc proto-oncogene are two factors that modulate the malignant potential of neuroblastoma. In cell lines, N-myc expression and malignancy are dramatically affected by cell differentiation state. Neuroblastic cells express high levels of N-myc and are highly tumorigenic whereas non-neuronal S-type cells from the same parental cell line contain markedly lower amounts of N-myc RNA/protein and are non-tumorigenic. The two cell types can arise from a third cell type, a more malignant I-type stem cell. Moreover, experimentally induced changes in cell phenotype can directly affect N- myc expression and transformation state. These observations form the basis for our central hypothesis: Cell differentiation state determined by lineage-specific as well as environmental factors, directly regulates N-myc expression and, thereby, the malignant potential of neuroblastoma cells. One lineage-specific factor regulating N-myc expression is HuD, a neuronal-specific RNA-binding protein. HuD appears in early neurogenesis and is expressed in all neurons. HuD binds to N-myc mRNA and, in antisense-HuD transfectants, its down-regulation results in reduced N-myc expression. The two aims of this proposal examine different aspects of this hypothesis. The focus of Aim #1 is to determine the mechanism(s) by which HuD interacts with N-myc mRNA and thereby regulates its expression and, as a corollary, whether loss of one HuD allele is a critical factor in the development of N-myc amplification and tumor aggressiveness. Studies proposed in Aim # 2 will establish whether the well characterized cell types found in human neuroblastoma cell lines (particularly I-type stem cells) are present in tumors and, if so, whether their presence correlates with tumor stage, aggressiveness, or site of origin. Knowledge gained of the differentiation status of cells in tumors with a favorable or unfavorable prognosis and of factors that can directly affect cellular differentiation, and thereby malignant potential, is highly likely to have important application to the treatment of neuroblastoma.